Heterochromatin is implicated in the regulation of diverse biological processes including recombination, chromosome segregation, transposon repression, and gene expression. As such, mutations in heterochromatin associated proteins are recurrently found in a variety of diseases, including Huntington's disease and numerous cancers. However, the role of these mutations in the progression of disease is poorly understood. A deeper understanding of heterochromatin regulation and the consequences of altered heterochromatin environments will be essential for understanding potential connections to disease. One potential mechanism of heterochromatin regulation is post-translational modification (PTM) of histones, including methylation of lysine nine on histone H3 (H3K9me). Several labs have attempted to study the role of H3K9me by mutating the enzymes that establish this modification. However, conclusions from their studies are limited because these enzymes have several non-histone substrates in addition to H3K9. Unfortunately, directly testing the role of H3K9 by mutating this residue to a non-modifiable amino acid has been unfeasible in higher eukaryotes due to the difficulty of engineering replacement histone genes. For this reason, although modification of H3K9 has been considered an important regulator of heterochromatin for more than a decade, the function of H3K9 in animals has never been directly tested. By using a Drosophila histone replacement platform recently established by our lab and collaborators, we will analyze the contribution of H3K9 to heterochromatin regulation by replacing endogenous histones with H3K9 mutant histones. The first aim of this study seeks to determine if mutating H3K9 results in altered heterochromatin structure by assessing recruitment of heterochromatin associated proteins and chromatin compaction. Given the ability of H3K9me to recruit heterochromatin factors we expect some genomic locations to lose heterochromatic proteins and consequently form a more open chromatin environment. We will test this idea by examining localization of Heterochromatin Protein 1 and heterochromatin associated histone PTMs via ChIP-seq and polytene chromosome cytology. Moreover, we will genetically assess heterochromatin formation using Position-Effect Variegation assays and explore genome-wide open chromatin profiles using FAIRE-seq to interrogate chromatin structure in H3K9 mutants. The second aim of this proposal will address the functional importance of H3K9 for two heterochromatin regulated processes, transposon repression and chromosome segregation. We hypothesize that a compromised heterochromatic environment will lead to de-repression of transposable elements and defects in chromosome segregation. We will test these hypotheses by performing quantitative PCR to analyze transposon expression and confocal microscopy to investigate chromosome segregation. Ultimately, we expect analysis of H3K9's role in regulating heterochromatin to augment our understanding of how altered chromatin structure may lead to disease.